The present invention relates to adenovirus vectors, more particularly, to propagation-defective adenovirus vectors.
The basis of gene therapy is to deliver a functional gene to tissues where the respective gene activity is missing or defective. Among the approaches to accomplishing gene therapy has been the use of recombinant viral vectors which have been genetically engineered to carry a desired transgene. These viral-based vectors have advantageous characteristics, such as the natural ability to infect the target tissue. However, implementation of existing viral vectors are impeded by several limitations as well.
For example, retrovirus-based vectors must integrate into the genome of the target tissue to allow for transgene expression (with the potential to activate resident oncogenes) while vector titers produced in such systems are significantly less than in some other systems. Because of the requirement for integration into the subject genome, the retrovirus vector can only be used to transduce actively dividing tissues. Further, many retroviruses have limited host tissue specificity and cannot be employed to transduce more than a few specific tissues of the subject.
Adenovirus vectors hold great promise for gene therapy. Adenovirus vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney, muscle (skeletal and cardiac), respiratory, and nervous system tissues. See, e.g., Askari et al., Gene Ther. 3:381-388 (1996); Barr et al., Gene Ther. 1:51-58 (1994); Engelhardt et al., Hum. Gene Ther. 4:759-769 (1993). Using transcomplementing packaging cell lines, first generation Adenovirus vectors can be grown and concentrated to high titers ( greater than 1013), which contributes to their ability to transduce large numbers of target cells after in vivo administration. Ragot, et al., Nature 361:647-50 (1993).
First generation adenovirus vectors also have a comparatively large carrying capacity (i.e., up to about 8.0 kb). The ability of first generation Adenovirus vectors to allow expression of transduced genes episomally for extended periods of time in immune-incompetent and sometimes immune-competent animals without the need for integration into the vector genome (Vincent, et al., Nat Genet. 5:130-34 (1993); Tripathy, et al., Nature Medicine 2:545-50 (1996)) allows them to transduce mitotically quiescent cells as well as actively dividing cells. Finally, live Adenovirus preparations have been used for the vaccination of military recruits, and Ad strains 2 and 5 (most commonly used for vector development) are not associated with severe disease.
Despite the advantages discussed above, first generation, E1-deleted adenovirus virus vectors are limited in potential therapeutic use for several reasons. First, due to the size of the E1 deletion and to physical virus packaging constraints, first generation adenovirus vectors are limited to carrying approximately 8.0 kb of transgene genetic material. While this compares favorably with other viral vector systems, it limits the usefulness of the vector where a larger transgene is required. Second, infection of the E1-deleted first generation vector into packaging cell lines leads to the generation of some replication competent adenovirus particles, because only a single recombination event between the E1 sequences resident in the packaging cell line and the adenovirus vector genome can generate a wild-type virus. Therefore, first-generation adenovirus vectors pose a significant threat of contamination of the adenovirus vector stocks with significant quantities of replication competent wild-type virus particles, which may result in toxic side effects if administered to a gene therapy subject.
The most difficult problem with adenovirus vectors is their inability to sustain long-term transgene expression, secondary to immune responses that eliminate virally transduced cells in immune-competent animals. Gilgenkrantz et al., Hum. Gene Ther. 6:1265-1274 (1995); Yang et al., J. Virol. 69:2004-2015 (1995); Yang et al., Proc. Natl. Acad. Sci. USA 91:4407-4411 (1994); Yang et al., J. Immunol. 155: 2565-2570 (1995). While immune responses have been demonstrated against the transgene-encoded protein product (Tripathy et al., Nat. Med. 2; 545-550 (1996)), it has also been demonstrated that adenovirus vector epitopes are major factors in triggering the host immune response. Gilgenkrantz et al., Hum. Gene Ther. 6:1265-1274 (1995); Yang et al., J. Viral. 70: 7209-7212 (1996). It has been repeatedly demonstrated that transgene such as the bacterial xcex2-galactosidase gene are highly immunogenic when transduced by adenovirus vectors, in contrast to other delivery systems (e.g., direct DNA injection or adeno-associated virus administration), where an immune response against the immunogenic transgene is lacking and transgene expression persists. Wolff et al., Hum. Mol. Genet. 1:363-369(1992); Xiao et al., J. Virol. 70:8098-8108 (1996).
In addition, E1xe2x88x92 vectors have also been reported to express the adenovirus early genes, undergo genome replication and express the L1-L5 encoded structural genes when utilized in vivo. E.g., Yang, et al., Immunity 1: 433-442 (1994). Because only a single recombination event is required to produce an entirely replication competent virus from the E-1 deletion, the exaggerated immune response may also be due in some instances to the contaminating presence of wild type adenovirus virus in the vector preparation. E.g., Rich, Hum. Gene. Ther. 4: 461-476 (1993). Either (or both) of these phenomena result in the production and presence of viral proteins in the transduced cells, possibly creating a higher antigenic profile than other gene therapy vector systems. The presence of these adenovirus viral gene products may contribute to the short duration of transgene expression in cells infected by first generation adenovirus vectors by accelerating the detection and elimination of adenovirus vector infected cells by the host immune system.
Accordingly, there remains a need in the art for improved adenovirus vector systems that address the limitations of existing systems.
The present invention provides novel deleted adenovirus vectors that provide advantages over existing xe2x80x9cfirst-generationxe2x80x9d adenovirus vectors. The deleted adenovirus vectors of the present invention may advantageously have an increased carrying capacity for heterologous nucleotide sequences, demonstrate lower levels of viral protein expression, induce fewer host immune responses, and/or exhibit increased stability and prolonged transgene expression when introduced into target cells.
The inventive adenovirus vectors carry one or more deletions in the IVa2, 100K, polymerase and/or preterminal protein sequences of the adenovirus genome. The adenoviruses may additionally contain other deletions, mutations or modifications as well. In particular preferred embodiments, the adenovirus genome is multiply deleted, i.e., carries two or more deletions therein. More preferably, there are deletions in two or more regions of the adenovirus genome (e.g., E1, E3, polymerase, 100K, IVa2, preterminal protein, etc.). At least one of the deletions in the adenovirus genome renders the adenovirus xe2x80x9cpropagation-defectivexe2x80x9d in that the virus cannot replicate and produce new virions in the absence of complementing function(s); preferably the vector carries multiple (two or more) deletions that result in a propagation-defective phenotype. When introduced into a trans-complementing cell that provides the deleted functions from the adenovirus genome, the deleted adenoviruses of the invention can produce a productive infection that results in the generation of new virus particles.
A further aspect of the present invention is methods for producing high-titers of the inventive deleted adenovirus vectors using packaging cells. Methods are also disclosed for producing the inventive deleted vectors using bacterial recombination and methods for producing xe2x80x9cguttedxe2x80x9d adenovirus vectors using deleted helper adenoviruses. Gutted adenovirus stocks according to the invention may exhibit increased stability and reduced viral protein expression from contaminating helper viruses as compared with previous preparations.
The inventive vectors can be administered to cells in vitro, e.g., to produce a protein/peptide or RNA of interest. In particular, a recombinant deleted adenovirus vector according to the invention may be administered to a cell in vitro, whereupon the cell expresses a heterologous nucleotide sequence. In particular embodiments, the nucleotide sequence encodes a protein or peptide (e.g., an enzyme) that is related to a metabolic disorder and/or a lysosomal or glycogen storage disorder. The expressed protein or peptide can be isolated, e.g., for protein replacement therapies.
In other embodiments, the recombinant adenovirus vectors of the invention may be administered to a cell in vitro and the cell administered to a subject, e.g., to produce an immunogenic or therapeutic response in the subject. In further alternative embodiments, the inventive deleted adenovirus vectors are administered directly to the subject. In particular, the present investigations have determined that intravenous administration to GAA-deficient animals of deleted adenovirus vectors of the invention carrying a GAA gene resulted in high-level transduction of liver cells and subsequent expression of the GAA transgene. Hepatic expression of GAA produced elevated plasma levels of GAA protein and significant reductions in glycogen levels in affected tissues.
The present invention also discloses methods of administering a recombinant deleted adenovirus vector of the invention into an organ or tissue, whereby a heterologous nucleotide sequence is expressed and the encoded protein/peptide or RNA is delivered to a different organ or tissue, e.g., to produce an immunogenic of therapeutic effect. For example, a nucleotide sequence encoding a foreign protein can be delivered to the liver, whereupon it is expressed and secreted into the circulatory system and delivered to target tissues (e.g., muscle). As a further alternative, the inventive adenovirus vectors can be introduced into the brain (e.g., by direct injection) for delivery of foreign proteins or nucleotide sequences to the central nervous system.
A further aspect of the invention is methods of expressing a protein or peptide in the liver for delivery to a distal tissue or organ (e.g., muscle tissue) to provide a therapeutic effect therein. Preferably, a deleted adenovirus of the invention is employed to introduce a nucleotide sequence encoding the protein or peptide into the liver. Also preferred are nucleotide sequences encoding proteins or peptides associated with a metabolic disorder, more preferably a lysosomal or glycogen storage disease (e.g., lysosomal acid xcex1-glucosidase). Further preferred are nucleotide sequences encoding lysosomal proteins or peptides.
These and other aspects of the invention are set forth in more detail in the description of the invention below.